Research of photosynthetic activity and physiological condition of plants is most frequently based on time dependent changes of chlorophyll fluorescence. Chlorophyll, as the key photoactive pigment is present in all photosynthetic organisms and is a photoactive chromophore of all reaction centres modifying radiant energy to biochemically usable form. Most of the absorbed energy is used by the photochemical system for transfer of electrons and protons from water to the products and processes necessary for CO2 fixation to sucrose molecular structure. The energy of photosynthetically active radiation (wavelength of 400-700 nm) is absorbed by antena pigments (chlorophylls and carotenoids) organized in light harvesting complexes of photosystem I and photosystem II, then it is transferred in the form of excitons to reaction centres of both the photosystems, where it is used in cascade of primary photochemical and non-photochemical processes for photosynthetic fixation of CO2.
Apart from photochemical reactions there are also processes of non-photochemical nature consuming excitation energy, which leads to thermal dissipation and chlorophyll fluorescence. In healthy plants about 80% of absorbed energy is consumed by photochemical reactions, only 3-5% represent chlorophyll fluorescence and the rest (15-17%) is transformed to heat. Increase of efficiency of one component, in which absorbed energy is transformed, leads to reduction in efficiency of at least one of the remaining components in accordance with the energy conservation law.
The principle of one of the common methods of investigation of photosynthesis or more precisely measurement of its efficiency is based on the premise that increase of excitation energy consumption by photochemical reactions leads to reduction of intensity of chlorophyll fluorescence, which is measured.
This measurement method belongs to a group of so called fluorimetric methods based on pulse amplitude modulation of a signal of the induced chlorophyll fluorescence. Fluorimetry enables us to determine and quantify the contribution of photochemical and non-photochemical processes to overall reduction of chlorophyll fluorescence obtained by assessment of records of so called chlorophyll fluorescence induction kinetics. Recently, photosynthetic activities of plants in photosynthesis research are mainly derived from these curves, measured under different external and internal conditions by means of various types of fluorimeters.
The published patent application DE 4427438 describes a method of research of plant photosynthetic system, where plants undergo phases of darkness and light, and after a dark phase they are illuminated with very short light pulses generated by a laser source with perpendicular course and induced fluorescent radiation is recorded. This is a non-destructive method, but it has a disadvantage that it studies the activity of photosystems I and II in plants and does not enable studying volume dynamics of plant tissues during photosynthesis.
Disadvantage of fluorimetric methods is generally based on the fact that they are not capable to measure directly the transformation of excitation energy to heat. Measurement of thermal signal of photosynthetically active samples thus has to be performed together with fluorescence to enable full investigation of the way how plants utilize the energy absorbed during photosynthesis.
A photoacoustic method is known for measurements of heat development and processes related to the exchange of gases in chloroplasts and in plant tissue. This method is based on the photoacoustic effect, i.e. a time recording of pressure modulation in samples, which includes acoustic waves induced by radiationless deexcitation of excited states of pigment molecules, development of gas (O2), fixation of gas (CO2), sample surface deformations etc., as a consequence of absorption of frequency modulated light. Acoustic waves in photosynthetically active samples are a result of thermal expansion in organic materials and ambient gases, photosynthetic oxygen development and CO2 uptake. Light absorption in photosynthetically active samples leads to both changes of dynamic pressure inside plant tissue cells, and volume changes of a sample as a whole.
Disadvantages of the photoacoustic method, also called photoacoustic spectroscopy, are mainly based on the fact that samples have to be measured in enclosed measurement cells, which is complicated, particularly with plants with regard to their size and positioning in a cell. This method is particularly advantageous for research of chloroplast suspensions, segments of leaves and tissues. The disadvantage is that a measured sample is separated from the mother plant, thus the measurement in so called in vivo status is impossible. The acquisition cost of the photoacoustic spectroscopy device is very high, as well.
Volume or more precisely dimensional changes may be generally measured for example by interferometric and holographic methods. The principle of exact interference measurement of distances is based on superposition of two coherent light waves (i.e. waves with a constant phase difference) which are directed at the same time to the same point. Evaluation of patterns appeared in the interference enables very exact measurement of length distances in the light wave path by means of an interferometer (e.g. so called Michelson interferometer).
The published patent application WO 01/27557 for example describes a system for interference deformation analysis in real time, which is modified for measurements in industrial environment. Phase differences of the measuring and the reference beams are recorded by means of a hologram.
Another published application WO 2005/001445 presents a method and a device for phase differential measurement of distance changes of small biological objects, particularly in the field of cell physiology and neurology, which cannot be examined in living conditions by for example X-ray. The system involves the method of optical interferometry or spectroscopy of diffuse radiation or their combination, and is intended for detection of changes in optical characteristics of the measured sample, not for measurement of the volume change dynamics in real time.
Another published application WO 2006/079013 similarly describes an interferometer with low-coherent beam for measurement of tissues of a biological sample, which directs a light beam to the first layer of the biological sample and receives the light reflected from this layer and then it directs the beam to a reflective device and receives the light reflected by the reflective device. The reflected light beams interfere and define the first phase. The second phase is defined similarly. The biological sample is evaluated upon the first phase and the second phase. The method is designed for measurement of atherosclerotic plaques in arterial system upon the index of refraction change in a sample environment.
Another known method and device for analysis of biological objects is described in a published patent application WO 2007/014 622, where the means of digital holographic microinterferometry are used, a 3-dimensional image of biological objects, particularly cells is captured, ad it is then evaluated, except others also from the point of view of volume and dimensional changes.
Holographic techniques cannot be applied on examination of photosynthetically active samples, as exposure of a sample to laser radiation would affect the sample photosynthesis already as a consequence of the measurement radiation itself, which is undesirable for the measurement. Holograms themselves moreover do not have sufficient resolution and radiation of larger object in vivo is technically complicated.
Application of known interference measurement methods based on laser measuring beams, applied in other fields of technology, to photosynthetically active samples is also problematic. Application of intensive high energy laser beams causes a damage and destruction of samples, for example a plant leaf, as a consequence of the fact that more than 80% of the measuring beam radiation is absorbed by the sample and damages its tissue. Photosynthetically active samples (e.g. leaves) have only low reflectivity (approx. 8-12%), which is why even interferometers with low energy laser beam cannot be used, as in this case the measuring beam would not be reflected with sufficient intensity and an interferogram of sufficient quality could not be obtained.
Application of interferometers known from other field of technology is then problematic for unsuitability of their detection and measurement parameters in the field of transmission of a laser interferential pattern with variable radiation intensity on the screen to an electric signal, which may be processed and evaluated as a sample dimension change.
A known interferometric length measurement method represents counting of pulses derived from the pass of a photodetector harmonic signal through a nod (zero), while the distance change is obtained as an order multiple of wavelength or its half.
Better resolution than ½ of the wavelength of the radiation applied is required for exact measurement of physiologically, particularly photosynthetically active samples. This implies exact measurement of relative values of the amplitude of detector signals, from which the signal phase is then determined, and dimension change can be determined to a fraction of wavelength.
Complication is caused by unstable intensity of laser beam, which changes as a consequence of time and temperature instabilities of a laser generator. The noise of the detected signal, which is mainly generated by self oscillations of mechanical scanning device, is another problem. Microphone effect occurs here, building vibrations are scanned and further signals disabling exact determination of relative amplitude of photodetector signals and subsequent phase determination. The measured signal itself is burdened by noise signal, containing also coincidental effects apart from harmonic and anharmonic components.